RESUMEN
BACKGROUND: Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity. METHODS: Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates. RESULTS: Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples. CONCLUSIONS: SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.
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Daño del ADN , Reparación del ADN , Análisis de la Célula Individual/métodos , Adulto , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Niño , Ciclina A2/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Haploinsuficiencia/efectos de los fármacos , Histonas/metabolismo , Recombinación Homóloga/efectos de los fármacos , Humanos , Mutágenos/toxicidad , Fosforilación/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reproducibilidad de los Resultados , TemozolomidaRESUMEN
The aim of this study was to assess the feasibility of applying the single cell network profiling (SCNP) assay to the examination of signaling networks in epithelial cancer cells, using bladder washings from 29 bladder cancer (BC) and 15 nonbladder cancer (NC) subjects. This report describes the methods we developed to detect rare epithelial cells (within the cells we collected from bladder washings), distinguish cancer cells from normal epithelial cells, and reproducibly quantify signaling within these low frequency cancer cells. Specifically, antibodies against CD45, cytokeratin, EpCAM, and cleaved-PARP (cPARP) were used to differentiate nonapoptotic epithelial cells from leukocytes, while measurements of DNA content to determine aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK at baseline and in response to pathway modulation; 66% (N = 19) of BC samples and 27% (N = 4) of NC samples met the "evaluable" criteria, i.e., at least 400,000 total cells available upon sample receipt with >2% of cells showing an epithelial phenotype. The majority of epithelial cells detected in BC samples were nonapoptotic and all signaling data were generated from identified cPARP negative cells. In four of 19 BC samples but in none of the NC specimens, SCNP assay identified epithelial cancer cells with a quantifiable increase in epidermal growth factor-induced p-AKT and p-ERK levels. Furthermore, preincubation with the PI3K inhibitor GDC-0941 reduced or completely inhibited basal and epidermal growth factor-induced p-AKT but, as expected, had no effect on p-ERK levels. This study demonstrates the feasibility of applying SCNP assay using multiparametric flow cytometry to the functional characterization of rare, bladder cancer cells collected from bladder washing. Following assay standardization, this method could potentially serve as a tool for disease characterization and drug development in bladder cancer and other solid tumors.
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Biomarcadores de Tumor/genética , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-akt/genética , Neoplasias de la Vejiga Urinaria/genética , Aneuploidia , Biomarcadores de Tumor/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/clasificación , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Citometría de Flujo/métodos , Humanos , Indazoles/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Análisis de la Célula Individual/métodos , Sulfonamidas/farmacología , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
PURPOSE: We conducted a single-center, dose-escalation study evaluating the safety, pharmacokinetics, and efficacy of epratuzumab, an anti-CD22 humanized monoclonal antibody, in patients with aggressive non-Hodgkin's lymphoma. EXPERIMENTAL DESIGN: Epratuzumab was administered once weekly for 4 weeks at 120-1000-mg/m2 doses to 56 patients [most (n = 35) with diffuse large B-cell lymphoma]. RESULTS: Patients were heavily pretreated (median, 4 prior therapies), 25% received prior high-dose chemotherapy with stem cell transplant, and 84% had bulky disease (> or =5 cm). Epratuzumab was well tolerated, with no dose-limiting toxicity. Most (95%) infusions were completed within 1 h. The mean serum half-life was 23.9 days. Across all dose levels and histologies, objective responses (ORs) were observed in five patients (10%; 95% confidence interval, 3-21%), including three complete responses. In patients with diffuse large B-cell lymphoma, 15% had ORs. Overall, 11 (20%) patients experienced some tumor mass reduction. Median duration of OR was 26.3 weeks, and median time to progression for responders was 35 weeks. Two responses are ongoing at > or =34 months, including one rituximab-refractory patient. CONCLUSIONS: These data demonstrate that epratuzumab has a good safety profile and exerts antitumor activity in aggressive non-Hodgkin's lymphoma at doses of > or =240 mg/m2, thus warranting further evaluation in this clinical setting.
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Anticuerpos Monoclonales/toxicidad , Linfoma no Hodgkin/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Moléculas de Adhesión Celular/inmunología , Terapia Combinada , Relación Dosis-Respuesta a Droga , Femenino , Estudios de Seguimiento , Semivida , Humanos , Infusiones Intravenosas , Lectinas/inmunología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/patología , Linfoma no Hodgkin/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Seguridad , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Factores de TiempoRESUMEN
INTRODUCTION: Antagonists of activin receptor signaling may be beneficial for cancer-related anemia and bone disease caused by malignancies such as multiple myeloma and solid tumors. AREAS COVERED: We review evidence of dysregulated signaling by activin receptor pathways in anemia, myeloma-associated osteolysis, and metastatic bone disease, as well as potential involvement in carcinogenesis. We then review properties of activin receptor antagonists in clinical development. EXPERT OPINION: Sotatercept is a novel receptor fusion protein that functions as a soluble trap to sequester ligands of activin receptor type IIA (ActRIIA). Preclinically, the murine version of sotatercept increased red blood cells (RBC) in a model of chemotherapy-induced anemia, inhibited tumor growth and metastasis, and exerted anabolic effects on bone in diverse models of multiple myeloma. Clinically, sotatercept increases RBC markedly in healthy volunteers and patients with multiple myeloma. With a rapid onset of action differing from erythropoietin, sotatercept is in clinical development as a potential first-in-class therapeutic for cancer-related anemia, including those characterized by ineffective erythropoiesis as in myelodysplastic syndromes. Anabolic bone activity in early clinical studies and potential antitumor effects make sotatercept a promising therapeutic candidate for multiple myeloma and malignant bone diseases. Antitumor activity has been observed preclinically with small-molecule inhibitors of transforming growth factor-ß receptor type I (ALK5) that also antagonize the closely related activin receptors ALK4 and ALK7. LY-2157299, the first such inhibitor to enter clinical studies, has shown an acceptable safety profile so far in patients with advanced cancer. Together, these data identify activin receptor antagonists as attractive therapeutic candidates for multiple diseases.
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Receptores de Activinas/antagonistas & inhibidores , Anemia/tratamiento farmacológico , Enfermedades Óseas/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Receptores de Activinas/metabolismo , Anemia/etiología , Anemia/metabolismo , Animales , Enfermedades Óseas/etiología , Enfermedades Óseas/metabolismo , Humanos , Neoplasias/complicaciones , Neoplasias/metabolismoRESUMEN
PURPOSE: 7-t-Butyldimethylsilyl-10-hydroxycamptothecin (AR-67) is a novel third generation camptothecin selected for development based on the blood stability of its pharmacologically active lactone form and its high potency in preclinical models. Here, we report the initial phase I experience with i.v. AR-67 in adults with refractory solid tumors. EXPERIMENTAL DESIGN AND METHODS: AR-67 was infused over 1 hour daily five times, every 21 days, using an accelerated titration trial design. Plasma was collected on the 1st and 4th day of cycle 1 to determine pharmacokinetic parameters. RESULTS: Twenty-six patients were treated at nine dosage levels (1.2-12.4 mg/m(2)/d). Dose-limiting toxicities were observed in five patients and consisted of grade 4 febrile neutropenia, grade 3 fatigue, and grade 4 thrombocytopenia. Common toxicities included leukopenia (23%), thrombocytopenia (15.4%), fatigue (15.4%), neutropenia (11.5%), and anemia (11.5%). No diarrhea was observed. The maximum tolerated dosage was 7.5 mg/m(2)/d. The lactone form was the predominant species in plasma (>87% of area under the plasma concentration-time curve) at all dosages. No drug accumulation was observed on day 4. Clearance was constant with increasing dosage and hematologic toxicities correlated with exposure (P < 0.001). A prolonged partial response was observed in one subject with non-small cell lung cancer. Stable disease was noted in patients with small cell lung cancer, non-small cell lung cancer, and duodenal cancer. CONCLUSIONS: AR-67 is a novel, blood-stable camptothecin with a predictable toxicity profile and linear pharmacokinetics. The recommended phase II dosage is 7.5 mg/m(2)/d five times every 21 days.